The rational development of new antineoplastic agents directed against tubulin, a protein critical for cell division, requires greater understanding of the interactions between the polypeptide subunits of tubulin and its two tightly bound guanine nucleotides. Interactions of ribose- and polyphosphate-modified GDP and GTP analogs with tubulin were examined in a microtubule-associated protein-dependent polymerization system. Although ribose-modified GTP analogs had a reduced affinity for tubulin, several of these nucleotides supported vigorous polymerization reactions by enhancing polymer nucleation, the rate-limiting step in microtubule assembly. Polyphosphate-modified analogs also had a reduced affinity for tubulin, except for guanosine 5'-0-(3-thiotriphosphate), which was a potent nucleotide inhibitor of tubulin polymerization and GTP hydrolysis. The effects of pH and the magnesium cation on the interactions of GDP and GTP with tubulin were examined in detail. In the course of preparing large amounts of microtubule-associated proteins, a protein component was isolated which caused microtubule bundle formation. Several new classes of antimitotic drugs were studied. These included combretastatin, a plant-derived natural product, and two groups of synthetic compounds, 6-benzyl-1, 3-benzodioxoles and 5,6-diarylpyridazin-3-ones. Efforts to separate the Alpha and Beta subunits of tubulin continued.